Cancer metastasis is the most deadly stage in cancer progression. Despite significant efforts over the past decades, it remains elusive why only a very small fraction of cancer cells is able to generate micrometastasis and metastatic colonization. Recently we have shown that tumor-repopulating cells (TRCs), a highly tumorigenic subpopulation of mouse melanoma cells, can be selected by being cultured and grown in 3D soft fibrin gels. Here we show that when injected into the yolk of a 2 day-post-fertilization (dpf) embryo of Tg (fli1:EGFP or kdrl:mCherry) zebrafish, TRCs are much more efficient in surviving and growing at various secondary sites to generate micrometastasis and metastatic colonization than control melanoma cells that are grown on rigid plastic. The metastasis of TRCs is dependent on the presence of Sox2, a self-renewal gene, and silencing Sox2 leads to the inhibition of TRC metastasis. High-resolution of 3D confocal images of the TRCs at the secondary sites show that extravasation and formation of micrometastases by TRCs are more efficient than by the control cells. Remarkably, efficient extravasation of TRCs in vivo and transmigration in vitro are determined by TRC deformability, as a result of low Cdc42 and high Sox2. Our findings suggest that tumor cell deformability is a key factor in controlling extravasation dynamics during metastasis.

DNase I hypersensitive sites (DHSs) provide important information on the presence of transcriptional regulatory elements and the state of chromatin in mammalian cells. Conventional DNase sequencing (DNase-seq) for genome-wide DHSs profiling is limited by the requirement of millions of cells. Here we report an ultrasensitive strategy, called single-cell DNase sequencing (scDNase-seq) for detection of genome-wide DHSs in single cells. We show that DHS patterns at the single-cell level are highly reproducible among individual cells. Among different single cells, highly expressed gene promoters and enhancers associated with multiple active histone modifications display constitutive DHS whereas chromatin regions with fewer histone modifications exhibit high variation of DHS. Furthermore, the single-cell DHSs predict enhancers that regulate cell-specific gene expression programs and the cell-to-cell variations of DHS are predictive of gene expression. Finally, we apply scDNase-seq to pools of tumour cells and pools of normal cells, dissected from formalin-fixed paraffin-embedded tissue slides from patients with thyroid cancer, and detect thousands of tumour-specific DHSs. Many of these DHSs are associated with promoters and enhancers critically involved in cancer development. Analysis of the DHS sequences uncovers one mutation (chr18: 52417839G>C) in the tumour cells of a patient with follicular thyroid carcinoma, which affects the binding of the tumour suppressor protein p53 and correlates with decreased expression of its target gene TXNL1. In conclusion, scDNase-seq can reliably detect DHSs in single cells, greatly extending the range of applications of DHS analysis both for basic and for translational research, and may provide critical information for personalized medicine.

Decellularized tendon hydrogel from human or porcine tendon has been manufactured and found to be capable of augmenting tendon repair in vivo. However, no studies have clarified the effect of decellularized tendon hydrogel upon stem cell behavior. In the present study, we developed a new decellularized tendon hydrogel (T-gel) from Macaca mulatta, and investigated the effect of T-gel on the proliferation, migration and tenogenic differentiation of Macaca mulatta tendon-derived stem cells (mTDSCs). The mTDSCs were first identified to have universal stem cell characteristics, including clonogenicity, expression of mesenchymal stem cell and embryonic stem cell markers, and multilineage differentiation potential. Decellularization of Macaca mulatta Achilles tendons was confirmed to be effective by histological staining and DNA quantification. The resultant T-gel exhibited highly porous structure or similar nanofibrous structure and approximately swelling ratio compared to the collagen gel (C-gel). Interestingly, stromal cell-derived factor-1 (SDF-1) and fibromodulin (Fmod) inherent in the native tendon extracellular matrix (ECM) microenvironment were retained and the values of SDF-1 and Fmod in the T-gel were significantly higher than those found in the C-gel. Compared with the C-gel, the T-gel was found to be cytocompatible with NIH-3T3 fibroblasts and displayed good histocompatibility when implanted into rat subcutaneous tissue. More importantly, it was demonstrated that the T-gel supported the proliferation of mTDSCs and significantly promoted the migration and tenogenic differentiation of mTDSCs compared to the C-gel. These findings indicated that the T-gel, with its retained nanofibrous structure and some bioactive factors of native tendon ECM microenvironment, represents a promising hydrogel for tendon regeneration.

Extracellular matrix (ECM) hydrogels are used as scaffolds to facilitate the repair and reconstruction of tissues. This study aimed to optimize the decellularization process of porcine skeletal muscle ECM and to formulate a matrix hydrogel scaffold. Five multi-step methods (methods A-E) were used to generate acellular ECM from porcine skeletal muscle [rinsing in SDS, trypsin, ethylenediaminetetraacetic acid (EDTA), Triton X-100 and/or sodium deoxycholate at 4-37°C]. The resulting ECM was evaluated using haematoxylin and eosin, 4-6-diamidino-2-phenylindole (DAPI) staining, and DNA quantification. Acellular matrix was dissolved in pepsin and gelled at 37°C. Hydrogel response to temperature was observed in vivo and in vitro. ECM components were assessed by Masson, Sirius red, and alcian blue staining, and total protein content. Acellular porcine skeletal muscle exhibited a uniform translucent white appearance. No intact nuclear residue was detected by haematoxylin and eosin staining, while DAPI staining showed a few nuclei in the matrixes produced by methods B, C, and D. Method A generated a gel that was too thin for gelation. However, the matrix obtained by rinsing in 0.2% trypsin/0.1% EDTA, 0.5% Triton X-100, and 1% Triton X-100/0.2% sodium deoxycholate was nuclei-free and produced a viscous solution that formed a structurally stable white jelly-like hydrogel. The residual DNA content of this solution was 49.37 ± 0.72 ng/mg, significantly less than in fresh skeletal muscle, and decreased to 19.22 ± 0.85 ng/mg after gelation (P < 0.05). The acellular matrix was rich in collagen and glycosaminoglycan, with a total protein concentration of 64.8 ± 6.9%. An acellular ECM hydrogel from porcine skeletal muscle was efficiently produced.

Mig6 is a feedback inhibitor that directly binds, inhibits and drives internalization of ErbB-family receptors. Mig6 selectively targets activated receptors. Here we found that the epidermal growth factor receptor (EGFR) phosphorylates Mig6 on Y394 and that this phosphorylation is primed by prior phosphorylation of an adjacent residue, Y395, by Src. Crystal structures of human EGFR-Mig6 complexes reveal the structural basis for enhanced phosphorylation of primed Mig6 and show how Mig6 rearranges after phosphorylation by EGFR to effectively irreversibly inhibit the same receptor that catalyzed its phosphorylation. This dual phosphorylation site allows Mig6 to inactivate EGFR in a manner that requires activation of the target receptor and that can be modulated by Src. Loss of Mig6 is a driving event in human cancer; analysis of 1,057 gliomas reveals frequent focal deletions of ERRFI1, the gene that encodes Mig6, in EGFR-amplified glioblastomas.

Keratinocyte growth factor 1 (KGF-1) has proven useful in the treatment of pathologies associated with dermal adnexae, liver, lung, and the gastrointestinal tract diseases. However, poor stability and short plasma half-life of the protein have restricted its therapeutic applications. While it is possible to improve the stability and extend the circulating half-life of recombinant human KGF-1 (rhKGF-1) using solution-phase PEGylation, such preparations have heterogeneous structures and often low specific activities due to multiple and/or uncontrolled PEGylation. In the present study, a novel solid-phase PEGylation strategy was employed to produce homogenous mono-PEGylated rhKGF-1. RhKGF-1 protein was immobilized on a Heparin-Sepharose column and then a site-selective PEGylation reaction was carried out by a reductive alkylation at the N-terminal amino acid of the protein. The mono-PEGylated rhKGF-1, which accounted for over 40% of the total rhKGF-1 used in the PEGylation reaction, was purified to homogeneity by SP Sepharose ion-exchange chromatography. Our biophysical and biochemical studies demonstrated that the solid-phase PEGylation significantly enhanced the in vitro and in vivo biostability without affecting the over all structure of the protein. Furthermore, pharmacokinetic analysis showed that modified rhKGF-1 had considerably longer plasma half-life than its intact counterpart. Our cell-based analysis showed that, similar to rhKGF-1, PEGylated rhKGF-1 induced proliferation in NIH 3T3 cells through the activation of MAPK/Erk pathway. Notably, PEGylated rhKGF-1 exhibited a greater hepatoprotection against CCl(4)-induced injury in rats compared to rhKGF-1.

Creating heterogeneous tissue constructs with an even cell distribution and robust mechanical strength remain important challenges to the success of in vivo tissue engineering. To address these issues, we are developing a scaffold sheet tissue engineering strategy consisting of thin (∼200 μm), strong, elastic, and porous crosslinked urethane-doped polyester (CUPE) scaffold sheets that are bonded together chemically or through cell culture. Suture retention of the tissue constructs (four sheets) fabricated by the scaffold sheet tissue engineering strategy is close to the surgical requirement (1.8 N) rendering their potential for immediate implantation without a need for long cell culture times. Cell culture results using 3T3 fibroblasts show that the scaffold sheets are bonded into a tissue construct via the extracellular matrix produced by the cells after 2 weeks of in vitro cell culture.

TCDD-inducible poly (ADP-ribose) polymerase (TiPARP) is a DNA repair enzyme with functions in energy metabolism, signal transduction, cell differentiation, and other biological processes, which may closely related to lipid metabolism and is highly expressed in adipose tissue. Adipose tissue can be divided into white adipose tissue (WAT) that stores energy and brown adipose tissue (BAT) that releases energy and generates heat. In the present study, we investigated whether TiPARP can affect adipogenesis in adipose tissue and thus participate in the development of obesity.

This study aimed to establish a high-fat diet (HFD)-fed obese mouse model and a cell culture model of insulin resistance (IR) in mature 3T3-L1 adipocytes. A dual-luciferase reporter assay (DLRA) was confirmed interaction between miR-27a and the 3'-untranslated region (UTR) of Peroxisome proliferator-activated receptor (PPAR)-γ. The inhibition of PPAR-γ expression by microRNA (miR)-27a in IR cells at both the protein and mRNA levels was confirmed by a mechanistic investigation. Moreover, the 3'-UTR of PPAR-γ was found to be a direct target of miR-27a, based on the DLRA. Furthermore, antagomiR-27a upregulated the activation of PI3K/Akt signaling and glucose transporter type 4 (GLUT4) expression at the protein and mRNA levels. Additionally, the PPAR inhibitor T0070907 repressed the insulin sensitivity upregulated by antagomiR-27a, which was accompanied by the inhibition of PPAR-γ expression and increased levels of AKT phosphorylation and GLUT4. The PI3K inhibitor wortmannin reduced miR-27a-induced increases in AKT phosphorylation, glucose uptake, and GLUT4. miR-27a is considered to be involved in the PPAR-γ-PI3K/AKT-GLUT4 signaling axis, thus leading to increased glucose uptake and decreased IR in HFD-fed mice and 3T3-L1 adipocytes. Therefore, miR-27a is a novel target for the treatment of IR in obesity and diabetes.

Prostate apoptosis response-4 (Par-4) is a tumor suppressor that induces apoptosis in cancer cells. However, the physiological function of Par-4 remains unknown. Here we show that conventional Par-4 knockout (Par-4-/-) mice and adipocyte-specific Par-4 knockout (AKO) mice, but not hepatocyte-specific Par-4 knockout mice, are obese with standard chow diet. Par-4-/- and AKO mice exhibit increased absorption and storage of fat in adipocytes. Mechanistically, Par-4 loss is associated with mdm2 downregulation and activation of p53. We identified complement factor c3 as a p53-regulated gene linked to fat storage in adipocytes. Par-4 re-expression in adipocytes or c3 deletion reversed the obese mouse phenotype. Moreover, obese human subjects showed lower expression of Par-4 relative to lean subjects, and in longitudinal studies, low baseline Par-4 levels denoted an increased risk of developing obesity later in life. These findings indicate that Par-4 suppresses p53 and its target c3 to regulate obesity.

Sustained c-Jun N-terminal kinase (JNK) activation plays a major role in drug-induced liver injury (DILI). Stress-responsive microRNA-31 (miR-31) has been implicated in regulating different cellular damage, and JNK activation could induce miR-31 expression. However, the regulatory role of miR-31 in DILI has not been studied previously. We aimed to investigate whether miR-31 could ameliorate DILI and ascertain potential molecular mechanism.

Angiotensin II (Ang II) plays a major role in the pathogenesis of cardiac fibrosis in hypertension. It is known that Ang II induces TGF-β1 expression. How transcription mediates Ang II-induced TGF-β1 expression, as well as its contribution to cardiac fibrosis, is unknown. We studied the role of Krüppel-like family transcription factors in Ang II-induced myofibroblast formation. We found that among the Krüppel-like family members, Krüppel-like factor 4 (Klf4) was the highest expressed form in isolated cardiac fibroblasts after Ang II treatment. Klf4 increased expression of α-SMA and collagen, as well as increased myofibroblast formation. ChIP assays showed that Klf4 specifically bound to the TGF-β1 promoter. Deletion and mutagenesis analysis showed that the sites at -184∼-180 bp and -45∼-41 bp in the TGF-β1 promoter were responsible for Klf4 transactivation of the TGF-β1 promoter. Our studies demonstrate that Klf4 plays a pivotal role in Ang II-induced cardiac myofibroblast differentiation and collagen synthesis through transcriptional upregulation of TGF-β1.

Histone deacetylase inhibitors (HDIs) represent a new class of anticancer drugs. Suberoylanilide hydroxamic acid (SAHA), the first HDI approved for the treatment of cutaneous T cell lymphoma (CTCL), is currently being tested in clinical trials for other cancers. However, SAHA has been ineffective against solid tumors in many clinical trials. A better understanding of molecular mechanisms of SAHA resistance may provide the basis for improved patient selection and the enhancement of clinical efficacy. Here we demonstrate that oncogenic K-ras contributes to SAHA resistance by upregulating HDAC6 and c-myc expression. We find that the high levels of HDAC6 expression are associated with activated K-ras mutant in colon cancer patients. And expressions of HDAC6 and c-myc are increased in fibroblasts transformed with activated K-ras. Surprisingly, we find that activated K-ras transformed cells are more resistant to SAHA inhibition on cell growth and anchorage-independent colony formation. We show that a K-ras inhibitor sensitizes K-ras mutated lung cancer cells to SAHA induced growth inhibition. We also find that mutant K-ras induces HDAC6 expression by a MAP kinase dependent pathway. Our study suggests that combined treatment with SAHA and K-ras inhibitors may represent an effective strategy to overcome SAHA resistance.

The H3K27 histone methyltransferase, Ezh2 (enhancer of zeste 2), is a Polycomb group protein that plays important roles in many biological processes including cellular differentiation, stem cell biology, and cancer development. Up-regulation of Ezh2 is observed in various human cancers consistent with its role in cell proliferation. Thus, understanding the regulation of Ezh2 may reveal how it contributes to the cellular proliferation process. Here, we demonstrate that Ezh2 can be regulated by the cyclin-dependent kinase, CDK1, which phosphorylates Ezh2 at threonines 345 and 487. Consistent with the cell cycle phase during which CDK1 exhibits peak activity, Ezh2 phosphorylation is enriched in cells arrested in mitosis when compared with S-phase. Phosphorylation of Thr-345 and Thr-487 promotes Ezh2 ubiquitination and subsequent degradation by the proteasome. Furthermore, expression of T345A/T487A confers a proliferative disadvantage when compared with cells expressing wild-type Ezh2, which suggests that phosphorylation of Ezh2 is important for cell proliferation. Collectively, these results establish a novel function for CDK1-mediated Ezh2 phosphorylation and provide a mechanism by which Ezh2 protein levels can be regulated in cells.

Fibroblast growth factor 21 (FGF21) is an important endogenous regulator involved in the regulation of glucose and lipid metabolism. FGF21 expression is strongly induced in animal and human subjects with metabolic diseases, but little is known about the molecular mechanism. Endoplasmic reticulum (ER) stress plays an essential role in metabolic homeostasis and is observed in numerous pathological processes, including type 2 diabetes, overweight, nonalcoholic fatty liver disease (NAFLD). In this study, we investigate the correlation between the expression of FGF21 and ER stress. We demonstrated that TG-induced ER stress directly regulated the expression and secretion of FGF21 in a dose- and time-dependent manner. FGF21 is the target gene for activating transcription factor 4 (ATF4) and CCAAT enhancer binding protein homologous protein (CHOP). Suppression of CHOP impaired the transcriptional activation of FGF21 by TG-induced ER stress in CHOP-/- mouse primary hepatocytes (MPH), and overexpression of ATF4 and CHOP resulted in FGF21 promoter activation to initiate the transcriptional programme. In mRNA stability assay, we indicated that ER stress increased the half-life of mRNA of FGF21 significantly. In conclusion, FGF21 expression is regulated by ER stress via ATF- and CHOP-dependent transcriptional mechanism and posttranscriptional mechanism, respectively.

Humanity has faced three recent outbreaks of novel betacoronaviruses, emphasizing the need to develop approaches that broadly target coronaviruses. Here, we identify 55 monoclonal antibodies from COVID-19 convalescent donors that bind diverse betacoronavirus spike proteins. Most antibodies targeted an S2 epitope that included the K814 residue and were non-neutralizing. However, 11 antibodies targeting the stem helix neutralized betacoronaviruses from different lineages. Eight antibodies in this group, including the six broadest and most potent neutralizers, were encoded by IGHV1-46 and IGKV3-20. Crystal structures of three antibodies of this class at 1.5-1.75-Å resolution revealed a conserved mode of binding. COV89-22 neutralized SARS-CoV-2 variants of concern including Omicron BA.4/5 and limited disease in Syrian hamsters. Collectively, these findings identify a class of IGHV1-46/IGKV3-20 antibodies that broadly neutralize betacoronaviruses by targeting the stem helix but indicate these antibodies constitute a small fraction of the broadly reactive antibody response to betacoronaviruses after SARS-CoV-2 infection.

Oncolytic virotherapy is being developed as a promising platform for cancer therapy due to its ability to lyse cancer cells in a tumor-specific manner. Vaccinia virus has been used as a live vaccine in the smallpox eradication program and now is being potential in cancer therapy with a great safety profile. Vaccinia strain Guang9 (VG9) is an attenuated Chinese vaccinia virus and its oncolytic efficacy has been evaluated in our previous study. To improve the tumor selectivity and oncolytic efficacy, we here developed a thymidine kinase (TK)-deleted vaccinia virus based on Guang9 strain. The viral replication, marker gene expression and cytotoxicity in various cell lines were evaluated; antitumor effects in vivo were assessed in multiple tumor models. In vitro, the TK-deleted vaccinia virus replicated rapidly, but the cytotoxicity varied in different cell lines. It was notably attenuated in normal cells and resting cells in vitro, while tumor-selectively replicated in vivo. Significant antitumor effects were observed both in murine melanoma tumor model and human hepatoma tumor model. It significantly inhibited the growth of subcutaneously implanted tumors and prolonged the survival of tumor-bearing mice. Collectively, TK-deleted vaccinia strain Guang9 is a promising constructive virus vector for tumor-directed gene therapy and will be a potential therapeutic strategy in cancer treatment.

In recent years, p53 was identified to regulate the expression of many miRNAs and was also regulated by miRNAs. In this paper, we found that miR-138 showed a pronounced increase after p53 activation in human non-small cell lung cancer (NSCLC) cells, which is mediated by p53 binding sites in the promoter region of its host gene, but this did not happen with rat and mouse cells. More interestingly, we found that p53 could be also regulated by miR-138 in mouse and rat cells, but not in the human NSCLC cells. Our results suggest the existence of species-specific differences of the regulations of miRNA against its targets and the regulations of miRNA itself by other proteins.

Biotin-labeled molecular probes, comprising specific regions of the SARS-CoV-2 spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. To develop such probes, we designed constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a C-terminal sequence targeted by biotin ligase. Probe regions included full-length spike ectodomain as well as various subregions, and we also designed mutants to eliminate recognition of the ACE2 receptor. Yields of biotin-labeled probes from transient transfection ranged from ~0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes were characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe was determined by cryo-electron microscopy. We also characterized antibody-binding specificities and cell-sorting capabilities of the biotinylated probes. Altogether, structure-based design coupled to efficient purification and biotinylation processes can thus enable streamlined development of SARS-CoV-2 spike-ectodomain probes.

Targeted protein degradation (TPD) has rapidly emerged as a therapeutic modality to eliminate previously undruggable proteins by repurposing the cell's endogenous protein degradation machinery. However, the susceptibility of proteins for targeting by TPD approaches, termed "degradability", is largely unknown. Here, we developed a machine learning model, model-free analysis of protein degradability (MAPD), to predict degradability from features intrinsic to protein targets. MAPD shows accurate performance in predicting kinases that are degradable by TPD compounds [with an area under the precision-recall curve (AUPRC) of 0.759 and an area under the receiver operating characteristic curve (AUROC) of 0.775] and is likely generalizable to independent non-kinase proteins. We found five features with statistical significance to achieve optimal prediction, with ubiquitination potential being the most predictive. By structural modeling, we found that E2-accessible ubiquitination sites, but not lysine residues in general, are particularly associated with kinase degradability. Finally, we extended MAPD predictions to the entire proteome to find 964 disease-causing proteins (including proteins encoded by 278 cancer genes) that may be tractable to TPD drug development.